Amyloid as a natural product

Amyloid fibrils, such as those found in Alzheimer's and the gelsolin amyloid diseases, result from the misassembly of peptides produced by either normal or aberrant intracellular proteolytic processing. A paper in this issue by Marks and colleagues (Berson et al., 2003) demonstrates that intra-melanosome fibrils are formed through normal biological proteolytic processing of an integral membrane protein. The resulting peptide fragment assembles into fibrils promoting the formation of melanin pigment granules. These results, along with the observation that amyloid fibril formation by bacteria is highly orchestrated, suggest that fibril formation is an evolutionary conserved biological pathway used to generate natural product nanostructures.     

Differential effects of two growth inhibitory K vitamin analogs on cell cycle regulating proteins in human hepatoma cells

A comparison was made between two K vitamin analogs. Growth in vitro of Hep G2 hepatoma cells was inhibited both by Compound 5 (Cpd 5), a recently synthesized thioalkyl analog of vitamin K or 2-(2-mercaptoethanol)-3-methyl-1, 4-naphthoquinone, as well as by synthetic vitamin K3 (menadione). Using synchronized Hep G2 hepatoma cells, the actions of both Cpd 5 and vitamin K3 on cell cycle regulating proteins were examined. Cpd 5 decreased the levels of cyclin D1, Cdk4, p16, p21 and cyclin B1. By contrast, VK3 only decreased the level of cyclin D1, but had no effect on the levels of Cdk4, p16 or p21. Interestingly, both VK3 and VK2 increased the levels of p21. The naturally occurring K vitamins had little effect on cell growth and none on the cyclins or Cdks. Amounts and activity of the G1/S phase controlling Cdc25A were measured. We found that Cpd 5 directly inhibited both Cdc25A activity and its protein expression, whereas VK3 did not. Thus, the main effects of Cpd 5 were on G1 and S phase proteins, especially Cdk4 and Cdc25A amounts in contrast to VK3. Computer docking studies of Cpd 5 and VK3 to Cdc25A phosphatase showed three binding sites. In the best conformation, Cpd 5 was found to be closer to the enzyme active site than VK3. These findings show that Cpd 5 represents a new class of anticancer agent, being a protein tyrosine phosphatase (PTP) antagonist, that binds to Cdc25A with suppression of its activity. Tumors expressing high levels of oncogenic Cdc25A phosphatase may thus be susceptible to the growth inhibitory activities of this class of compound.     

A microplate assay specific for the enzyme aggrecanase

We have identified a 41-residue peptide, bracketing the aggrecanase cleavage site of aggrecan, that serves as a specific substrate for this enzyme family. Biotinylation of the peptide allowed its immobilization onto streptavidin-coated plates. Aggrecanase-mediated hydrolysis resulted in an immobilized product that reveals an N-terminal neoepitope, recognized by the specific antibody BC-3. This assay is highly specific for aggrecanases; MMPs were inactive in this assay. Reduction of the peptide size below 30 amino acids resulted in a significant diminution of activity. Using the immobilized 41-residue peptide as a substrate, we have developed a 96-well microplate-based assay that can be conveniently used for high-throughput screening of samples for aggrecanase activity and for discovery of inhibitors of aggrecanase activity.     

ASC is an activating adaptor for NF-kappa B and caspase-8-dependent apoptosis

ASC is a pro-apoptotic protein containing a pyrin domain (PD) and a caspase-recruitment domain (CARD). A previous study suggests that ASC interacts with Ipaf, a member of the Apaf-1/Nod1 protein family. However, the functional relevance of the interaction has not been determined. Here, we report that co-expression of ASC with Ipaf or oligomerization of ASC induces both apoptosis and NF-kappa B activation. Apoptosis induced through ASC was inhibited by a mutant form of Caspase-8 but not by that of Caspase-1. The PD of ASC physically interacted with Caspase-8 as well as with pyrin, the familial Mediterranean fever gene product. Caspase-8 deficiency rescued mouse fibroblasts from apoptosis induced by ASC oligomerization. Pyrin disrupted the interaction between ASC and Caspase-8, and inhibited both apoptosis and NF-kappa B activation induced by ASC. These findings suggest that ASC is a mediator of NF-kappa B activation and Caspase-8-dependent apoptosis in an Ipaf signaling pathway.     

Cys10 mixed disulfides make transthyretin more amyloidogenic under mildly acidic conditions

Conservative mutation of transthyretin's surface residues can predispose an individual to familial amyloidosis by dramatically changing the energetics of misfolding. Senile systemic amyloidosis (SSA), however, cannot be explained in this fashion because wild-type (WT) transthyretin (TTR) misfolds and misassembles into amyloid. Since various modifications of the SH functionality of Cys10 have been reported in humans, we sought to understand the extent to which these modifications alter the stability and amyloidosis of WT TTR as a possible explanation for SSA. Homotetrameric Cys10 TTR variants, including TTR-Cys, TTR-GSH, TTR-CysGly, and S-sulfonated TTR, were chemically synthesized starting with WT TTR. The TTR-Cys, TTR-GSH, and TTR-CysGly isoforms are more amyloidogenic than WT at the higher end of the acidic pH range (pH 4.4-5.0), and they are similarly destabilized relative to WT TTR toward urea denaturation. They exhibit rates of urea-mediated tetramer dissociation (pH 7) and MeOH-facilitated fibril formation similar to those of WT TTR. Under mildly acidic conditions (pH 4.8), the amyloidogenesis rates of the mixed disulfide TTR variants are much faster than the WT rate. S-Sulfonated TTR is less amyloidogenic and forms fibrils more slowly than WT under acidic conditions, yet it exhibits a stability and rates of tetramer dissociation similar to those of WT TTR when subjected to urea denaturation. Conversion of the Cys10 SH group to a mixed disulfide with the amino acid Cys, the CysGly peptide, or glutathione increases amyloidogenicity and the amyloidogenesis rate above pH 4.6, conditions under which TTR probably forms fibrils in humans. Hence, these modifications may play an important role in human amyloidosis.     

Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid anandamide

The endocannabinoid arachidonylethanolamide (AEA, anandamide) is an endogenous ligand for the cannabinoid receptors and has been shown to be oxygenated by cyclooxygenase-2 (COX-2). We examined the structural requirements for COX-mediated, AEA oxygenation using a number of substrate analogues and site-directed mutants of COX-2. Fourteen AEA analogues were synthesized and tested as COX substrates. These studies identified the hydroxyl moiety of AEA as a critical determinant in the ability of COX enzymes to effect robust endocannabinoid oxygenation. In addition, these studies suggest that subtle structural modifications of AEA analogues near the ethanolamide moiety can result in pronounced changes in their ability to serve as COX-2 substrates. Site-directed mutagenesis studies have permitted the development of a model of AEA binding within the COX-2 active site. As with arachidonic acid, the omega-terminus of AEA binds in a hydrophobic alcove near the top of the COX-2 active site. The polar ethanolamide moiety of AEA, like the carboxylate of arachidonate, interacts with Arg-120 at the bottom of the COX-2 active site. Mutation of Tyr-385 prevents AEA oxygenation, suggesting that, as in the case of other COX substrates, AEA metabolism is initiated by Tyr-385-mediated hydrogen abstraction. Thus, AEA binds within the COX-2 active site in a conformation roughly similar to that of arachidonic acid. However, important differences have been identified that account for the isoform selectivity of AEA oxygenation. Importantly, the COX-2 side pocket and Arg-513 in particular are critical determinants of the ability of COX-2 to efficiently generate prostaglandin H(2) ethanolamide. The reduced efficiency of COX-1-mediated, AEA oxygenation can thus be explained by the absence of an arginine residue at position 513 in this isoform. Mutational analysis of Leu-531, an amino acid located directly across from the COX-2 side pocket, suggests that AEA is shifted away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Coupled with earlier observations with the endocannabinoid 2-arachidonylglycerol, these results indicate that one possible function of the highly conserved COX-2 active site side pocket is to promote endocannabinoid oxygenation.     

Convenient preparation of L-arabino-hexos-5-ulose derivatives from lactose

2,6-di-O-benzyl- (9), 2-O-benzyl-3,4-O-isopropylidene- (19), and 2-O-benzyl-6-O-m-chlorobenzoyl-L-arabino-hexos-5-ulose (20) have been prepared using 4'-deoxy-4'-eno- and 6'-deoxy-5'-eno lactose dimethyl acetal derivatives 7 and 14 as key intermediates. The synthesis of enol ethers 7 and 14 has been performed with good yields by base-promoted elimination of acetone or p-toluenesulfonic acid from 2',6'-di-O-benzyl-, and 6'-O-p-toluenesulfonyl-2,3:5,6:3',4'-tri-O-isopropylidenelactose dimethyl acetal, respectively. The epoxidation with MCPBA of 7 and 14 in methanol or dichloromethane furnishes C-5'-methoxy and C-5'-m-chlorobenzoyloxy derivatives, easily transformed with good yields into L-arabino 5-ketoaldohexoses 9, 19 and 20.     

Polar targeting of DivIVA in Bacillus subtilis is not directly dependent on FtsZ or PBP 2B

DivIVA is involved in Bacillus subtilis cell division and is located at the cell poles. Previous experiments suggested that the cell division proteins FtsZ and PBP 2B are required for polar targeting of DivIVA. By using outgrowing spores, we show that DivIVA accumulates at the cell poles independent of the presence of FtsZ or PBP 2B.     

Insulin inhibits PDGF-directed VSMC migration via NO/ cGMP increase of MKP-1 and its inactivation of MAPKs

In this study, we examined the roleof insulin in the control of vascular smooth muscle cell (VSMC)migration in the normal vasculature. Platelet-derived growth factor(PDGF) increased VSMC migration, which was inhibited by pretreatmentwith insulin in a dose-dependent manner. Insulin also caused a 60%decrease in PDGF-stimulated mitogen-activated protein kinase (MAPK)phosphorylation and activation. Insulin inhibition of MAPK wasaccompanied by a rapid induction of MAPK phosphatase (MKP-1), whichinactivates MAPKs by dephosphorylation. Pretreatment with inhibitors ofthe nitric oxide (NO)/cGMP pathway, blocked insulin-induced MKP-1 expression and restored PDGF-stimulated MAPK activation and migration. In contrast, adenoviral infection of VSMCs with MKP-1 or cGMP-dependent protein kinase I (cGK I), the downstream effector of cGMPsignaling, blocked the activation of MAPK and prevented PDGF-directedVSMC migration. Expression of antisense MKP-1 RNA prevented insulin's inhibitory effect and restored PDGF-directed VSMC migration and MAPKphosphorylation. We conclude that insulin inhibition of VSMC migrationmay be mediated in part by NO/cGMP/cGK I induction of MKP-1 andconsequent inactivation of MAPKs.